Available Assays

A 100% mutation detection rate has been reported in individuals with a germline mutation in the VHL gene using a combination of quantitative Southern blot analysis and DNA sequence analysis.8 Quantitative Southern blot analysis is performed by digestion of high-molecular-weight genomic DNA with restriction enzymes such as Eco RI and Ase I that cut at the 5' and 3' boundaries of the gene to yield a fragment approximately 9.7kb in size. Use of a double digest results in more efficient transfer of the gene fragment and avoids false negatives that occasionally result from digestion with Eco RI alone. Blots are hybridized with a VHL gene-specific probe (g7) to check for partial gene deletions and a probe for a two-copy gene (such as beta globin) to check for complete gene deletions. Partial gene deletions are detected as a decrease in intensity of the VHL gene band and the appearance of a band of altered mobility in the patient's sample (see Figure 23-1, lane 6). Complete gene deletions are detected as a decrease in intensity of the VHL gene band relative to the beta globin band in the patient compared to control samples (see Figure 23-1, lanes 3 and 5). Partial and complete gene deletions account

Figure 23-1. Quantitative Southern blot analysis for partial or complete deletions in the VHL gene. Genomic DNA (5 |g) is digested with Eco RI and Ase I. Digestion products are separated by agarose gel electrophoresis and transferred to a nitrocellulose membrane by capillary blotting. The filter is hybridized to random primer-labeled probes specific for the VHL gene (g7) and the beta globin gene (as an internal standard for DNA loading). Normal controls (lanes 1,2,4, and 7) exhibit a single intense VHL gene band. Patients with a partial deletion in the VHL gene exhibit a less-intense VHL band and a band of altered mobility (lane 6). Patients with a complete deletion of the VHL gene exhibit a band of decreased intensity (lanes 3 and 5) compared to controls when equal amounts of DNA are loaded on the gel. (Reprinted with permission from Chernoff A, Kaspar-cova V, Linehan WM, Stolle CA. Molecular analysis of the von Hippel-Lindau disease gene. In: Methods in Molecular Medicine Serles,vol 53: Renal Cancer: Methods and Protocols. Mydlo JH,ed.Humana Press Inc., Totowa, NJ; 2001, p. 193-216.

for approximately 28% of all cases of VHLD, with complete deletions occurring in about 3% to 5% of patients. Recently, a real-time quantitative polymerase chain reaction (PCR) assay has been described for the rapid detection of complete and partial deletions in the VHL gene.9

PCR amplification and DNA sequence analysis of exons 1 to 3 of the VHL gene (including the adjacent splice donor and acceptor sequences) will detect all disease-causing point mutations. DNA sequence analysis is typically performed on an automated DNA sequencer using fluores-cently labeled dideoxy terminator nucleotides. Both single base changes and small insertions or deletions may be detected as the appearance of two bases in any given posi-tion(s) of the DNA sequencing chromatogram (see Figure 23-2, panels b and c). A mutation-scanning method such as conformational sensitive gel electrophoresis (CSGE) may be used to identify mutation-containing exons prior to DNA sequence analysis. CSGE involves denaturing and reannealing PCR products to form heteroduplexes and homoduplexes that then are separated on a polyacrylamide gel. Exons exhibiting shifts (see Figure 23-3, lanes 4,7, and 10) are sequenced to identify the specific mutation.

(frameshift) results in a chromatogram with two bases in every position after the mutation (panel b). A single base change is recognized by the appearance of two bases in one position (panel c). Note that the height of the normal peak is generally reduced when a mutation is present in the same position. (Reprinted with permission from Chernoff et al.,Mol-ecular analysis of the von Hippel-Lindau disease gene. In: Mydlo JH, ed. Renal Cancer:Methods and Protocols. Humana Press Inc; 2001.)

(frameshift) results in a chromatogram with two bases in every position after the mutation (panel b). A single base change is recognized by the appearance of two bases in one position (panel c). Note that the height of the normal peak is generally reduced when a mutation is present in the same position. (Reprinted with permission from Chernoff et al.,Mol-ecular analysis of the von Hippel-Lindau disease gene. In: Mydlo JH, ed. Renal Cancer:Methods and Protocols. Humana Press Inc; 2001.)

Exon 1 Exon 2__Exon 3

Exon 1 Exon 2__Exon 3

Figure 23-3. Conformational sensitive gel electrophoresis (CSGE) of the three VHL exons. PCR products from exons 1 to 3 of the VHL gene are denatured and reannealed. The mobility of heteroduplexes containing a normal and a mutant strand are "shifted" (lanes 4,7, and 10), indicating the presence of a mismatched base (mutation) in the PCR product, compared to homoduplexes containing two normal strands after separation on a polyacrylamide gel. (Reprinted with permission from Chernoff et al., Molecular analysis of the von Hippel-Lindau disease gene. In: Mydlo JH,ed. Renal Cancer:Methods and Protocols. Humana Press Inc; 2001.)

Figure 23-3. Conformational sensitive gel electrophoresis (CSGE) of the three VHL exons. PCR products from exons 1 to 3 of the VHL gene are denatured and reannealed. The mobility of heteroduplexes containing a normal and a mutant strand are "shifted" (lanes 4,7, and 10), indicating the presence of a mismatched base (mutation) in the PCR product, compared to homoduplexes containing two normal strands after separation on a polyacrylamide gel. (Reprinted with permission from Chernoff et al., Molecular analysis of the von Hippel-Lindau disease gene. In: Mydlo JH,ed. Renal Cancer:Methods and Protocols. Humana Press Inc; 2001.)